Faculty Bibliography

Recently, the ubiquitin proteasome system (UPS) has matured as a drug discovery arena, largely on the strength of the proven clinical activity of the proteasome inhibitor Velcade in multiple myeloma. Ubiquitin ligases tag cellular proteins, such as oncogenes and tumor suppressors, with ubiquitin. Once tagged, these proteins are degraded by the proteasome. The specificity of this degradation system for particular substrates lies with the E3 component of the ubiquitin ligase system (ubiquitin is transferred from an E1 enzyme to an E2 enzyme and finally, thanks to an E3 enzyme, directly to a specific substrate). The clinical effectiveness of Velcade (as it theoretically should inhibit the output of all ubiquitin ligases active in the cell simultaneously) suggests that modulating specific ubiquitin ligases could result in an even better therapeutic ratio. At present, the only ubiquitin ligase leads that have been reported inhibit the degradation of p53 by Mdm2, but these have not yet been developed into clinical therapeutics. In this review, we discuss the biological rationale, assays, genomics, proteomics and three-dimensional structures pertaining to key targets within the UPS (SCFSkp2 and APC/C) in order to assess their drug development potential. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com)

In silico docking of a chemical library with the ligand binding domain (LBD) of thyroid hormone nuclear receptor-alpha (TRalpha) suggested that farnesyl pyrophosphate (FPP), a key intermediate in cholesterol synthesis and protein farnesylation, might function as an agonist. Surprisingly, addition of FPP to cells activated TR as well as the classical steroid hormone receptors but not peroxisome proliferative activating receptors, farnesol X receptor, liver x receptor, or several orphan nuclear receptors whose ligands are unknown. FPP enhanced receptor-coactivator binding in vitro and in vivo and elevation of FPP levels in cells by squalene synthetase or farnesyl transferase inhibitors leads to activation. The FPP effect was blocked by selective receptor antagonists, and in silico docking with 143 nuclear receptor LBD structures revealed that FPP only docked with the agonist conformation of those receptors activated by FPP. Our results suggest that certain nuclear receptors maintain a common structural feature that may reflect an action of FPP on an ancient nuclear receptor or that FPP could function as a ligand for one of the many orphan nuclear receptors whose ligands have not yet been identified. This finding also has potential interesting implications which may, in part, explain the pleotropic effects of statins as well as certain actions of farnesylation inhibitors in cells

An actomyosin motor located underneath the plasma membrane drives motility and host-cell invasion of apicomplexan parasites such as Plasmodium falciparum and Plasmodium vivax, the causative agents of malaria. Aldolase connects the motor actin filaments to transmembrane adhesive proteins of the thrombospondin-related anonymous protein (TRAP) family and transduces the motor force across the parasite surface. The TRAP-aldolase interaction is a distinctive and critical trait of host hepatocyte invasion by Plasmodium sporozoites, with a likely similar interaction crucial for erythrocyte invasion by merozoites. Here, we describe 2.4-A and 2.7-A structures of P. falciparum aldolase (PfAldo) obtained from crystals grown in the presence of the C-terminal hexapeptide of TRAP from Plasmodium berghei. The indole ring of the critical penultimate Trp-residue of TRAP fits snugly into a newly formed hydrophobic pocket, which is exclusively delimited by hydrophilic residues: two arginines, one glutamate, and one glutamine. Comparison with the unliganded PfAldo structure shows that the two arginines adopt new side-chain rotamers, whereas a 25-residue subdomain, forming a helix-loop-helix unit, shifts upon binding the TRAP-tail. The structural data are in agreement with decreased TRAP binding after mutagenesis of PfAldo residues in and near the induced TRAP-binding pocket. Remarkably, the TRAP- and actin-binding sites of PfAldo seem to overlap, suggesting that both the plasticity of the aldolase active-site region and the multimeric nature of the enzyme are crucial for its intriguing nonenzymatic function in the invasion machinery of the malaria parasite

The third variable region (V3) of the HIV-1 surface glycoprotein, gp120, plays a central role in the interaction of the virus envelope with the cell surface chemokine receptors, triggering membrane fusion and virus entry into human lymphocytes and macrophages. The CXCR4 and CCR5 chemokine receptors are used by 'X4-tropic' and 'R5-tropic' viruses, respectively. Recently, the crown of the V3 loop was shown to bear a close structural homology to the beta2-beta3 loop in the CXC and CC chemokines, the natural ligands of CXCR4 and CCR5, respectively. This homology can serve as the foundation for 3D molecular modeling of the V3 loops from primary isolates whose coreceptor usage was experimentally defined. The modeling revealed a charged 'patch' on the surface of V3 that correlates with coreceptor usage. This V3 surface patch is positively charged in X4-tropic viruses and negatively charged or neutral in R5-tropic viruses, and is formed by two amino acids, at position 11 and at position 24 or 25; amino acids 11 and 24 or 11 and 25 contact each other in 3D space. Residues at positions 11 and 25 were known previously to influence coreceptor usage, and the charge of the residues at these two positions is often used to predict viral tropism. However, we found that the predictive value of using the charge of residues 11, 24, and 25 to identify X4 or R5 tropism was improved over using only the charge of residues 11 and 25. Thus, the data suggest a new ' 11/24/25 rule' : a positively charged amino acid at position 11, 24, or 25 defines X4; otherwise R5. This rule gave an overall predictive value of 94% for 217 viruses whose tropism had been determined experimentally as either X4 or R5. The results have additional implications for the design of HIV therapeutics, vaccines, and strategies for monitoring disease progression

A complex molecular motor empowers substrate-dependent motility and host cell invasion in malaria parasites. The interaction between aldolase and the transmembrane adhesin thrombospondin-related anonymous protein (TRAP) transduces the motor force across the parasite surface. Here, we analyzed this interaction by using state-of-the-art flexible docking. Besides algorithms to account for induced fit in the side-chains of the Plasmodium falciparum aldolase (PfAldo) structure, we used additional in silico receptors modeled upon crystallographic structures of evolutionarily related aldolases to incorporate enzyme backbone flexibility, and to overcome structure inaccuracies due to the relatively low resolution (3.0 A) of the genuine PfAldo structure. Our results indicate that, in spite of multiple intermolecular contacts, only the six C-terminal residues of the TRAP cytoplasmic tail bind in an ordered manner to PfAldo. This portion of TRAP targets the PfAldo active site, with its n-1 Trp residue, which is essential for this interaction, buried within the PfAldo catalytic pocket. Docking of a TRAP peptide bearing a Trp to Ala mutation rendered the lower energy configurations either bound weakly outside the active site or not bound to PfAldo at all. The position of the bound TRAP peptide, and particularly the close proximity between the carbonyl of its n-2 Asp residue and the experimentally determined position of the phosphate-6 group of fructose 1,6-phosphate bound to mammalian aldolases, predicts an inhibitory effect of TRAP on catalysis. Enzymatic and TRAP-binding assays using mutant PfAldo molecules strongly support the overall structural model. These results might provide the initial framework for the identification of novel antiparasitic compounds

The known sequences of HIV-1 viruses have been categorized into subtypes based on the phylogenetic partitioning of their env and gag gene sequences. The env gene encodes the protein gp120, which contains five sequence- variable regions (V1 to V5), of which the V3 loop is of central importance to viral infectivity. The V3 loop consensus sequences of HIV-1 subtype A and C viruses are similar, and more similar to one another than the V3 consensus sequences of any other two HIV-1 subtypes. However, using a position-specific statistical comparison, we found that the V3 region of these two subtypes is statistically distinct (p = approximately 0.0). (The p-value calculated to the lowest limit of representation on the computer used to run the calculation. This lowest limit was 10(16). Although theoretically a p-value cannot be equal to 0.0, the p-value for the comparisons in question can be intuitively considered to be extremely small, or approximately 0.0.)

The transcriptional activity of the androgen receptor (AR) is modulated by interactions with coregulatory molecules. It has been proposed that aberrant interactions between AR and its coregulators may contribute to diseases related to AR activity, such as prostate cancer and androgen insensitivity syndrome (AIS); however, evidence linking abnormal receptor:cofactor interactions to disease is scant. The Androgen Receptor Trapped clone-27 (ART-27) is a recently identified AR N-terminal coactivator that is associated with AR-mediated growth inhibition. Here we analyze a number of naturally occurring AR mutations identified in prostate cancer and AIS for their ability to affect AR response to ART-27. Although the vast majority of AR mutations appeared capable of increased activation in response to ART-27, an AR mutation identified in prostate cancer (AR P340L) and AIS (AR E2K) show reduced transcriptional responses to ART-27, whereas their response to the p160 class of coactivators was not diminished. Relative to the wild-type receptor, less ART-27 protein associated with the AR E2K substitution, consistent with reduced transcriptional response. Surprisingly, more ART-27 associated with AR P340L, despite the fact that the mutation decreased transcriptional activation in response to ART-27. Our findings suggest that aberrant AR-coactivator association interferes with normal ART-27 coactivator function resulting in suppression of AR activity and may contribute to the pathogenesis of diseases related to alterations in AR activity, such as prostate cancer and AIS

The unique mechanism of the SCF ubiquitin ligase poses a challenge to drug discovery. A central enzymatic small molecule-binding active site is not evident in this multisubunit protein enzyme, as is the case with kinases or proteases. Instead, the SCF ligase seems to accomplish ubiquitylation through a series of cooperative movements dependent on the protein interfaces between its components and its substrate. Activity-modulating small molecules, therefore, need to interact with these protein interfaces. The three-dimensional structure of these interfaces may be the key asset in determining their suitability for small molecule binding. Computational tools and a systematic approach described in detail here can assess the 'druggability' of an SCF ligase before the investment of effort in high-throughput screening (HTS), structure-based drug design (SBBD), or virtual library screening (VLS)

Ubiquitin ligases are well suited to regulate molecular networks that operate on a post-translational timescale. The F-box family of proteins - which are the substrate-recognition components of the Skp1-Cul1-F-box-protein (SCF) ubiquitin ligase - are important players in many mammalian functions. Here we explore a unifying and structurally detailed view of SCF-mediated proteolytic control of cellular processes that has been revealed by recent studies